Cardiovascular disease (CVD) is currently the leading cause of death and is predicted to be the number one cause of disability worldwide by 2030 (World Health Organization 2014). In the United States, approximately 1 of every 3 deaths in 2010 was due to CVD. Ischemic heart disease and stroke were the leading primary causes of premature death. The overall economic impact of CVD was estimated to be $445 billion annually in the U.S. (2010), and is expected to increase substantially in future decades. More effective diagnostic tools and therapies are necessary to limit the growing burden of CVD in the U.S. and worldwide, particularly the diseases which manifest in unwanted clotting within the arteries of the heart or brain.
A major contributor to acute cardiovascular events and sudden deaths is the development of atherosclerotic plaques, a progressive thickening of the arterial wall due to the accumulation of cholesterol. Rupture of atherosclerotic plaques can form thrombi that occlude blood flow, potentially leading to a life-threatening event. Thrombi occurring in the coronary artery can lead to a heart attack, and in cerebral arteries can lead to ischemic stroke.
The early detection and treatment of CVD is vital to assess the risk of vulnerable plaque leading to an acute cardiovascular event. However, screening for vulnerable atherosclerotic plaque using current imaging modalities poses specific challenges. Direct visualization using noninvasive imaging methods, e.g. carotid ultrasound, cardiovascular computed tomography, magnetic resonance imaging, and positron emission tomography, are preferable for early diagnosis of vulnerable atherosclerotic plaque in high-risk patients. For example, carotid ultrasound with measurement of the intima-media thickness within the artery wall offers a way to diagnose the extent of subclinical atherosclerotic vascular disease, assess risk, and may offer a means to identify disease progression and monitor the effectiveness of preventive therapies. The use of microbubble based ultrasound contrast agents as a complementary tool to enhance vascular ultrasound imaging, known as contrast-enhanced ultrasound imaging, is emerging as an important method in facilitating the detection and characterization of atherosclerotic disease.
The use of microbubbles as ultrasound contrast agents (UCAs) in vascular imaging is well established. Most commercially available UCAs consist of gas-filled microbubbles which have mean diameters between 1-5 μm and are encapsulated with a protein, polymer, or lipid shell. Albunex® (GE Healthcare) was the first UCA approved by the U.S. Food and Drug Administration and consisted of an air-filled microbubble encapsulated by an albumin shell. Second generation UCAs such as Optison® (GE Healthcare), Definity® (Lantheus Medical Imaging) and Lumason® (Bracco Diagnostics, Inc.) contain high-molecular-weight gases (e.g. C3F8 and SF6 respectively), which have lower solubility in blood and thus increase the lifetime of the microbubbles in circulation. The low density and high compressibility of the gas core in UCAs enables efficient ultrasound scattering. Thus, the injected agents are acoustically active, or echogenic, and function as intravascular tracers which can be visualized using ultrasound.
In addition to traditional contrast-enhanced ultrasound imaging, there has been recent interest in advancing the applications of UCAs for molecular imaging of atherosclerosis. Molecular imaging techniques with targeted UCAs are being used increasingly for noninvasive diagnosis of inflammation, thrombus, and neovascularization. Targeted microbubble agents are also being developed for controlled drug-delivery applications and have been vigorously promoted for therapeutic applications in the treatment of CVD. Targeted UCAs are functionalized by engineering the gas-encapsulating shell to contain molecules that adhere to cells which express disease-specific markers (e.g., aminoacids) on the membrane. Phospholipid-shelled UCAs are of particular interest for this purpose, because they can be targeted to molecular components of disease by attaching specific ligands to the surface.
Phospholipid-shelled UCAs represent one type of UCA that is currently available for clinical use. The lipid molecules employed in the formulations are typically amphiphilic molecules which spontaneously form micelle structures that can encapsulate and stabilize a gas microbubble in an aqueous environment. The lipids are surface-active molecules (surfactants) which orient their hydrophilic polar groups outside towards the surrounding aqueous medium and their hydrophobic tails inside away from the water, stabilizing the microbubble and largely preventing the gas from escaping the encapsulation. Lipid-based ultrasound contrast agents such as Definity® and Lumason® (which was recently approved for clinical use in the U.S. but has been marketed as SonoVue® in Europe and Asia since 2001) are commercially available for diagnostic applications. MicroMarker® (VisualSonics, Toronto, Canada; Bracco Research SA, Geneva, Switzerland) and Targestar® (Targeson Inc., San Diego, Calif., USA) are examples of targeted phospholipid-shelled UCAs currently available for pre-clinical investigational use.
A more recent formulation in the broad category of phospholipid-shelled UCAs, known as echogenic liposomes (ELIP), has been developed which encapsulates both a gas and an aqueous phase (Alkan-Onyuksel et al. 1996; Huang et al. 2001). Standard liposomes are characterized by a phospholipid bilayer shell which encapsulates an aqueous compartment. ELIP are said to be echogenic because they contain a gas microbubble that is highly reflective to ultrasound waves at low intensities. The exact location of the entrapped gas pockets in ELIP has not been fully ascertained, and may be due to gas pockets stabilized by lipid monolayers within the liposome, or within the lipid bilayer shell. Various proposed schematics of echogenic liposomes have been put forth in the literature. Two possible models of an echogenic liposome (ELIP) with an outer phospholipid bilayer and a lipid monolayer shell surrounding a gas bubble are presented in FIG. 9A and FIG. 9B. The models are qualitatively similar, the only difference being the ratio of the total particle volume occupied by the internal gas bubble. With respect to mechanism of gas entrapment, previous studies have suggested that the freeze-drying procedure is key to the generation of defects in the lipid bilayers such that upon rehydration, they fuse and trap small amounts of gas. After reconstitution in an aqueous suspension, the phospholipid molecules are known to stabilize the gas core by imparting low surface tension and high mechanical stability.
At low pressure amplitudes, ELIP have been utilized as an UCA to enhance image quality. Further, at high ultrasound pressure amplitudes, the microbubbles can be forced to expand and may cause the liposome membrane to rupture, thereby releasing the encapsulated gas or drug for a potential therapeutic effect. ELIP are therefore known as theragnostic agents because they can be used for both diagnostic and therapeutic purposes.
ELIP formulations differ from current commercially available contrast agents primarily in size, shell material, and gas content. Previous studies have shown that ELIP range in size from 70 nm to several microns. ELIP formulations typically contain 3 or 4 phospholipid components and also include a small amount of cholesterol, which acts to increase membrane rigidity. The gas content can be air, which is more soluble in blood than high molecular weight gases. However, unlike commercially available UCAs, ELIP with optimized lipid formulations have been shown to be both echogenic and stable under physiologic conditions for tens of minutes. Compared to commercially available UCAs, ELIP are unique in that they can function as diagnostic imaging contrast agents and can also serve as therapeutic drug carriers.
Targetable drug-delivery systems represent a fast developing area of nanotechnology and are expected to have a dramatic impact on medicine in the future. Many nano-scale drug carriers, such as liposomes, micelles, and polymer nanocapsules, have been developed or are under development for encapsulation and delivery of therapeutic drugs. Liposomes are a convenient, biologically compatible vehicle for administration of poorly soluble drugs, and are among the first generation of nano-scale drug delivery systems to be approved for clinical use and known as “nanomedicines” (Moghimi et al. 2005).
Gregoriadis and Ryman (1971) were the first to report on the use of liposomes as drug carriers for directed delivery. The authors hypothesized that encapsulation of enzymes within the aqueous inner compartment of liposomes would aid in directing the payload to a particular tissue and alleviate some of the problems associated with immunological response to the proteins in circulation. They found that liposomes remain largely intact during circulation and are cleared by lysosomes in the liver (and to a lesser extent in the spleen). Since then, liposome based drug-delivery systems have been developed using chemotherapeutic agents for cancer therapy, thromolytic agents, and genes, in addition to enzymes.
Most of the currently approved liposome formulations represent a basic form of nanomedicine involving a passive targeting and drug release process known as the enhanced permeability and retention (EPR) effect. This approach relies on extravasation and accumulation of the liposome-encapsulated drug at the target site, and is particularly suited for cancer therapy applications due to the enhanced vascular permeability of tumors compared with normal tissue. Because tumors are highly vascularized and often lack effective lymphatic drainage, liposomes tend to accumulate in tumors much more than they do in normal tissues, resulting in increased drug uptake in these regions. Although EPR is a rudimentary passive targeting method, it is a key reason liposomes are currently the most widely used drug nanocarrier in cancer therapy. To realize the drug delivery potential of liposomes for other applications fully, however, it is important to develop agents with an active triggering mechanism that allows the drug to be delivered in a more controlled fashion. Echogenic liposomes, by virtue of their ability to encapsulate gas as well as therapeutic drugs, offer such a possibility.
Recently, ultrasound has been investigated as a method to trigger enhanced drug delivery within the human vasculature. The potential of ultrasound to control drug delivery spatially and temporally in a non-invasive manner is broadly appealing. Ultrasound-mediated drug delivery (UMDD) has been demonstrated in a number of tissue beds, for example the blood-brain barrier, cardiac tissue, prostate, and large arteries.
Acoustic cavitation is one physical mechanism that is hypothesized to influence UMDD. Cavitation as used herein refers to nonlinear bubble activity that can occur near vessel walls within the vasculature upon ultrasound exposure, which can exert mechanical stress on nearby cells and junctions. Mechanical stress can disturb the barriers to drug delivery such as endothelial tight junctions or phospholipid membranes, via transient permeabilization. In vivo, cavitation can be nucleated at moderate acoustic pressure amplitudes (<0.5 MPa) by ultrasound contrast agents (UCAs).
Conventional strategies for studying ultrasound-mediated drug release and delivery in vitro and ex vivo involve various techniques, from optical to electrophysiological. Optical techniques, such as fluorescence or luminescence, rely on the native optical properties of the therapeutic, or conjugation of tracer molecules. Electrophysiological approaches, such as voltage-clamp techniques, directly assess the changes in membrane potential provoked during UMDD, but often require isolated cells cultured in vitro, where cellular processes can vary drastically from in vivo conditions. In vivo animal models of UMDD provide relevant bioeffect information, yet are costly and subject to considerable inter-subject variability. The ability to detect and monitor the response of intact, isolated vascular tissue in real time would constitute a significant advancement is UMDD.
Nitric Oxide (NO) is a gas molecule that dynamically modulates the physiological functions of the cardiovascular system, which include relaxation of vascular smooth muscle, inhibition of platelet aggregation, and regulation of immune responses. Because a reduced NO level has been implicated in the onset and progression of various disease states, NO is expected to provide therapeutic benefits in the treatment of cardiovascular diseases, such as essential hypertension, stroke, coronary artery disease, atherosclerosis, platelet aggregation after percutaneous transluminal coronary angioplasty, and ischemia/reperfusion injury. To date, pharmacologically active compounds that can release NO within the body, such as organic nitrates and sodium nitroprusside, have been used as therapeutic agents, but their efficacy is significantly limited by their rapid NO release, poor distribution to the target site, toxicity, and induction of tolerance. Attenuation of nitric oxide production in the etiology of atherosclerosis progression and diabetic vascular disease further highlights the need for novel therapeutic nitric oxide modulation and delivery strategies. Effective delivery of bioactive NO to target cardiovascular tissue remains a compelling need in the art.
Isolated tissue bath perfusion systems have been used extensively to characterize contractility changes induced by a therapeutic in a variety of muscular tissue beds including gastric, peripheral vascular, and cardiovascular. In these systems, dose-dependent changes in active muscular tension can be characterized in response to vasorelaxing agents such as bradykinin, sodium nitropursside, nitroglycerine, and NO. Development of an isolated tissue bath model to investigate UMMD could provide relevant, real time quantitative data on the drug release and delivery profiles triggered by ultrasound.